Encapsulated pharmacological and cosmetic agents have several advantages over non-encapsulated agents. The bioavailability of the encapsulated agent can be improved, the active agent can be protected from degradation in a finished formulation, and delayed or slow, sustained release of the active agent is possible if the agent is encapsulated.
Active pharmacological or cosmetic agents can be encapsulated by incorporation into microspheres made of biocompatible, biodegradable natural polymers. A microsphere is a substantially spherical particle with a diameter in the μm range. Biocompatible, biodegradable natural polymers suitable for use in microspheres include collagen, fibrin, fibronectin, albumin, gelatin, starch, and hyaluronic acid.
Hyaluronic acid (HA) is a viscoelastic biopolymer composed of repeating disaccharide units of N-acetyl-D-glucosamine (GlcNAc) and D-glucuronic acid (GlcUA). Sodium hyaluronate is the predominate form of hyaluronic acid at physiological pH. Sodium hyaluronate and hyaluronic acid are collectively referred to as hyaluronan. Hyaluronan molecules have differing molecular weights due to the fact that the number of repeating disaccharide units in each molecule is variable. Sodium hyaluronate occurs naturally in cellular surfaces, in the basic extracellular substances of the connective tissues of vertebrates, in the synovial fluid of joints, in the vitreous humor of the eye, and in the tissue of umbilical cord. Sodium hyaluronate acts as a regulator of viscosity, tissue hydration, lubrication, and repair, and is involved in cell mobility, cell differentiation, wound healing, and cancer metastasis. Hyaluronan solutions and cross-linked hyaluronan gels can be used as drug delivery systems (U.S. Pat. No. 5,128,326). A drug can be dispersed in a hyaluronan solution, and a cross-linked hyaluronan gel can serve as a macromolecular cage in which a drug substance can be dispersed. In this manner, a hyaluronan gel or solution can serve as a vehicle that allows for the slow release of a drug that is incorporated into the gel or solution.
A large number of sodium hyaluronate derivatives have been synthesized by esterification of the carboxyl group of the D-glucuronic acid moiety of the sodium hyaluronate. (U.S. Pat. No. 4,851,521; Goei, L., et al., Pharmaceutical Research 6(9) S94 (1989); Ghezzo, E., et al., International Journal of Pharmaceutics 87: 21-29 (1992)). Ester derivatives of sodium hyaluronate have been used to form microspheres (Kyyrönen, K., et al., International Journal of Pharmacetuics 80: 161-169 (1992)); Ghezzo, E., et al., International Journal of Pharmaceutics 87:21-29 (1992); Richardson, J. L., et al., International Journal of Pharmaceutics 115: 9-15 (1995); Benedetti, L., et al., Biotechnology and Bioengineering 53: 232-237 (1997); U.S. Pat. No. 5,690,954). In addition, cross-linked esters of hyaluronic acid have been used to form microspheres, which can be incorporated into a bioabsorbable matrix to form wound implant materials (U.S. Pat. No. 5,766,631).
Sodium hyaluronate can also be derivatized by covalent attachment of hydrazides at carboxyl groups of glucuronic acid moieties (Pouyani, T. & Prestwich, G. D., Bioconjugate Chemistry 5:339-347 (1994); Vercruysse, K. P., et al., Bioconjugate Chemistry 8:686-694 (1997); U.S. Pat. No. 5,652,347, U.S. Pat. No. 5,616,568). Hyaluronate functionalized with hydrazide has a pendant hydrazide group that allows for subsequent coupling and crosslinking reactions (Pouyani, T. & Prestwich, G. D., Bioconjugate Chemistry 5:339-347 (1994); Vercruysse, K. P., et al., Bioconjugate Chemistry 8:686-694 (1997); U.S. Pat. No. 5,652,347, U.S. Pat. No. 5,616,568).
Various techniques have been used to produce microspheres made of sodium hyaluronate derivatives. A spray drying process has been used to prepare microspheres composed of sodium hyaluronate esters (Kyyronen, K., et al., International Journal of Pharmaceutics 80: 161-169 (1992)). An emulsion and solvent extraction procedure has been used to prepare microspheres composed of water-insoluble sodium hyaluronate esters (Ghezzo, E., et al., International Journal of Pharmaceutics 87: 21-29(1992)). In this approach, an emulsion was prepared in which the internal phase was a 6% w/v hyaluronate ester solution in dimethylsulphoxide (DMSO) containing the agents to be encapsulated, and the external phase consisted of mineral oil and 0.5% w/v of a surfactant. The inner phase was added to the outer phase with continuous stirring. Extraction with ethyl acetate proceeded until microspheres were formed. The microspheres were washed extensively with n-hexane and dried under a vacuum. Complete separation of the residual solvents could not be achieved with this emulsion/solvent extraction method, however, and a relevant percentage of liquid was retained within the microspheres. The presence of DMSO, ethyl acetate, and n-hexane in a composition that is to be administered to humans or animals is undesirable.
Attempts have been made to use a rapid expansion of supercritical solutions (RESS) process and a supercritical antisolvent process (SAS) to prepare microspheres composed of sodium hyaluronate benzylic esters (Bendetti, L., et al., Biotechnology and Bioengineering 53: 232-237 (1997)). In the RESS process, a supercritical fluid is used to solubilize a nonvolatile solute. The resulting solution is highly compressible and a sharp decrease in the solvent density, which can be obtained by a relatively small change in pressure, leads to a large decrease in the solubility of the solute. Solute nucleation, triggered by a sudden pressure decrease, is performed in media in which high supersaturation ratios are uniformly reached, which can lead to the formation of microparticles.
The SAS process is performed by first dissolving a solid of interest in an organic liquid (DMSO). Then, the supercritical fluid (CO2), which is not able to dissolve the solid but is completely miscible with the liquid, is added to the solution in order to precipitate the solute. The SAS continuous process is performed in the critical region of the CO2-DMSO system. The SAS batch process involves a low pressure gradient value and a uniform distribution of the antisolvent in the liquid.
The RESS process could not be used to prepare hyaluronic acid benzylic ester microspheres because the solubility of the hyaluronic acid ester in CO2 was too low (Bendetti, L., et al., Biotechnology and Bioengineering 53: 232-237 (1997)). Using the SAS continuous process, appreciable amounts of solute were produced, but the particles formed were not regular in shape and morphology, and agglomerate structures were obtained. When the SAS process was carried out in a batch mode, microspheres of the sodium hyaluronate ester were obtained that had an average diameter of 0.4 μm and a narrow particle size distribution.
To date, the art has not provided a hydrophilic hyaluronic acid microsphere, e.g., that may be useful for delivery of a pharmaceutical or cosmetic.